AAV Vector Engineering Based On Directed Evolution And Rational Design

Adeno-associated virus(AAV)is a type of non-enveloped virus with icosahedral structure and single-stranded DNA of the Parvoviridae.As an efficient and safe gene delivery vector with broad tropism,it is widely used in gene therapy,neural circuit tracing,in vivo imaging,gene editing,and development and evaluation of neurological disease models.However,AAV still has shortcomings such as poor transduction ability for specific tissues or cells and poor penetration of tissue barriers such as the blood-brain barrier.In this thesis,based on systematical understanding of the features in different natural serotypes of AAV,we designed and built mutation library for AAV capsid structural gene Cap,employing the directed evolution and rational design strategies,as a proof of the library screening paradigm,we took "acquiring AAV mutants that efficiently cross the blood-brain barrier" as the initial screening target.First,the natural serotype AAV is the basis of the optimization and modification work,in order to provide reference standards for the subsequent works,we packaged 13 natural serotypes of AAVs,and explored the infection characteristics at the cellular level,we found that AAV6,AAV2 and AAV 1 showed better infection efficiency on various cell lines,especially AAV6 shows better infection characteristics for glial cell lines,which suggests that AAV6 can be used as the preferred template serotype in the modification of AAV vectors targeting glial cells in the future.Second,in order to optimize the plasmid system for loading the Cap gene library and preparing the AAV virus library,five plasmid-packaging schemes for AAV library production were designed and the plasmids were constructed and compared experimentally.It was found that the "pAAV-ITR-P41-lox Cap" core plasmid combined with "Rep2-AAP helper"plasmid had the most robust packaging feature,so this scheme was used in packaging of subsequent mutant libraries.Then,based on the methods of directed evolution and rational design,taking the abundance of the library into account,we also introduced rationalization factors for the screening target of "acquiring AAV mutants that efficiently cross the blood-brain barrier" in order to reduce the redundancy of the gene library.Cap gene library was prepared and inserted into the previously verified "pAAV-ITR-P41-lox Cap" core plasmid,generating the pAAV-Shuffling plasmid library based on the DNA shuffling method,the pAAV-PD library(approximately 11810 mutants)based on the peptide display method,and 19 kinds of pAAV-ZH plasmid series based on rational design.Finally,the plasmid library was transfected into HEK293T cell line to produce the AAV library,the AAV-PD library(3.14×1012 VG/ml)and 19 AAV-ZH mutants.Comparing the packaging titers of different mutants,it was found that the longer the foreign peptide inserted in the AAV capsid,the lower the efficiency for the virion-packaging.According to the preliminary evaluation results at the cellular level,ZH05 and ZH06 showed infectivity toward glioma cells(U87),which might be of value for further charactorization.In this thesis a systematic paradigm for constructing AAV mutant libraries based on directed evolution technologies such as DNA shuffling and peptide display was tentatively established,several AAV mutants and one AAV virus library that could be used for subsequent screening were obtained.These works laid the foundation for further systematic modification of various viral vectors in the future.